scholarly journals A PCR-based Assay for Detection of Alternaria radicina on Carrot Seed

Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 18-23 ◽  
Author(s):  
B. M. Pryor ◽  
R. L. Gilbertson
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Skim Milk ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Silica Matrix ◽  
Chain Reaction ◽  
Carrot Seed ◽  
Natural Infestation ◽  
Polymerase Chain ◽  
Rapd Fragment

A pair of polymerase chain reaction (PCR) primers was developed based upon the sequence of a cloned random amplified polymorphic DNA (RAPD) fragment of Alternaria radicina, and a PCR-based seed assay was developed for the detection of A. radicina from infested carrot seed. The seed assay involved a 5-day incubation step, in which seed was maintained under high humidity conditions in order to increase fungal biomass. Seed was then incubated with lysis buffer, extracted with phenol-chloroform, and DNA was recovered using a silica matrix. PCR amplification of the target A. radicina DNA sequence was enhanced by the addition of skim milk to the PCR reaction mixture. With this PCR-based seed assay, A. radicina was detected from carrot seed lots with natural infestation rates as low as 0.3%. In seed lots prepared by mixing known amounts of A. radicina-infested seed with noninfested seed, this assay allowed for the detection of the pathogen from lots with infestation rates as low as 0.1%.

Molecular Differentiation of Two Strains of the Parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae) Using Specific PCR Primers

2004 ◽  
Vol 39 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Yu Cheng Zhu ◽  
Alan K. Dowdy ◽  
James E. Baker
Keyword(s):  
Dna Markers ◽  
Natural Populations ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Dna Fragments ◽  
Chain Reaction ◽  
Specific Primers ◽  
Specific Pcr ◽  
Anisopteromalus Calandrae ◽  
Polymerase Chain

Two strains (Sav and Bam) of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae) showed different sensitivity to organophosphate insecticides. By using polymerase chain reaction (PCR) and DNA sequencing, we demonstrated clear molecular difference between these two strains. DNA markers that are specific for the Bam strain were developed, and PCR-generated DNA fragments were cloned and sequenced. Two DNA fragments unique to the Bam strain contained 365 and 584 nucleotides. A pair of specific primers was designed from each fragment. PCR-amplification of the DNA from individual wasps generated fragments of the expected sizes only in the Bam strain. Studies conducted on F1 and F2 hybrids produced from crossing and backcrossing between resistant and susceptible strains indicated that these DNA markers are located on mitochondria and inherited exclusively maternally. Probes developed from these fragments may be used in assessing genetic information of natural populations and in studies on physiological or biochemical differences between the strains of this beneficial insect.

Differentiation of isolates in the genus Steinernema (Nematoda: Steinernematidae).by random amplified polymorphic DNA fragments and morphological characters

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J. Liu ◽  
R. E. Berry
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Nematode Species ◽  
Morphological Characters ◽  
Dna Fragments ◽  
Undescribed Species ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Polymerase Chain

SUMMARYWe combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.

scholarly journals Development of Polymerase Chain Reaction Primers Highly Specific for Xanthomonas albilineans, the Causal Bacterium of Sugarcane Leaf Scald Disease

Plant Disease ◽  
1999 ◽  
Vol 83 (3) ◽  
pp. 218-222 ◽  
Author(s):  
Y.-B. Pan ◽  
M. P. Grisham ◽  
D. M. Burner ◽  
B. L. Legendre ◽  
Q. Wei
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Multiple Sequence ◽  
Leaf Scald ◽  
Specific Pcr ◽  
Polymerase Chain ◽  
Xanthomonas Albilineans

New primers were developed that greatly improved the specificity of the polymerase chain reaction (PCR) protocol for Xanthomonas albilineans, the causal agent of sugarcane leaf scald disease. Length-polymorphic PCR products, amplified under the current PCR protocol from the 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) of X. albilineans and three unidentified sugarcane saprophytic bacterial species, were cloned and sequenced. Fourteen other nonredundant ITS sequences retrieved from the database were highly homologous to the sequence of X. albilineans. Two X. albilineans-specific PCR primers, namely, PGBL1 (5′ CTT TGG GTC TGT AGC TCA GG) and PGBL2 (5′ GCC TCA AGG TCA TAT TCA GC), were designed based on a multiple sequence alignment among these 18 sequences. These two primers permitted specific PCR amplification of a 288-bp DNA product from all 71 diverse X. albilineans strains tested. No amplification product was observed from any other bacterial species tested, including the three unidentified sugarcane saprophytes. The new PCR protocol has been routinely used to detect the leaf scald pathogen from infected sugarcane tissues.

Influence of Growth in a Food Medium on the Detection of Escherichia coli O157:H7 by Polymerase Chain Reaction

2002 ◽  
Vol 65 (11) ◽  
pp. 1775-1779 ◽  
Author(s):  
JOHN L. McKILLIP ◽  
LEE-ANN JAYKUS ◽  
MARYANNE DRAKE
Keyword(s):  
Cell Density ◽  
Skim Milk ◽  
Pcr Amplification ◽  
Amplification Efficiency ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Dna Yield ◽  
Polymerase Chain ◽  
Food Medium

The effects of storage time and growth in broth culture and in a food medium on the efficiency of Escherichia coli O157: H7 DNA extraction and on the sensitivity of polymerase chain reaction (PCR) detection of E. coli O157:H7 were investigated. Detection limits were evaluated with dilution series PCR targeting the slt-II gene. The relationship between cell density and DNA yield was generally log-linear for pure cultures of E. coli O157:H7. When the bacteria were suspended in skim milk at a density of 106 CFU/ml, held at 4°C, and sampled at 24-h intervals, cell density, total DNA yield, and PCR detection limits remained stable throughout the 96-h storage period. However, when E. coli O157:H7 was grown in skim milk to a final cell density of 106 CFU/ml, PCR amplification efficiency was drastically reduced, although overall DNA yields from these samples were consistent with those for the samples in which E. coli O157:H7 growth was static over 96 h of storage at 4°C. This result is most likely due to poor DNA purity, which was consistently observed when DNA was extracted from food matrices in which the pathogen was grown rather than stored. The results of this investigation underscore the likelihood that multiple components may drastically affect DNA extraction and PCR amplification efficiency in the detection of pathogens in the food matrix. It is clear that before nucleic acid amplification technologies are widely applied to food systems, it would be prudent to test their efficacy in multiple food matrices and under conditions in which the bacterial population is both static and actively growing.

scholarly journals Characterization of Xanthomonas axonopodis pv. phaseoli isolates

2008 ◽  
Vol 34 (3) ◽  
pp. 228-231 ◽  
Author(s):  
Willian Mário de Carvalho Nunes ◽  
Maria Júlia Corazza ◽  
Silvana Aparecida Crestes Dias de Souza ◽  
Siu Mui Tsai ◽  
Eiko Eurya Kuramae
Keyword(s):  
Xanthomonas Campestris ◽  
Random Amplified Polymorphic Dna ◽  
Hypersensitive Reaction ◽  
Chain Reaction ◽  
Xanthomonas Axonopodis ◽  
Paulo State ◽  
Total Dna ◽  
Polymerase Chain ◽  
Bacterial Plant Pathogen

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

Detection ofErwinia amylovorain plant material using novel polymerase chain reaction (PCR) primers

2001 ◽  
Vol 29 (1) ◽  
pp. 35-43 ◽  
Author(s):  
R. K. Taylor ◽  
P. J. Guilford ◽  
R. G. Clark ◽  
C. N. Hale ◽  
R. L. S. Forster
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Amplification of human beta-actin gene by the reverse transcriptase-polymerase chain reaction: implications for assessment of RNA from formalin-fixed, paraffin-embedded material.

1996 ◽  
Vol 44 (10) ◽  
pp. 1205-1207 ◽  
Author(s):  
A Dakhama ◽  
V Macek ◽  
J C Hogg ◽  
R G Hegele
Keyword(s):  
Polymerase Chain Reaction ◽  
Pcr Amplification ◽  
Housekeeping Gene ◽  
Chain Reaction ◽  
Viral Genomes ◽  
Negative Results ◽  
Beta Actin ◽  
Target Nucleic Acid ◽  
Formalin Fixed Paraffin Embedded ◽  
Polymerase Chain

The polymerase chain reaction (PCR) is a powerful method that allows enzymatic amplification of rate target nucleic acid sequences. It has been applied to the amplification of viral genomes from paraffin-embedded pathology specimens. However, interpretation of negative results requires amplification of a housekeeping gene such as beta-actin. In the present study we used specific oligonucleotide primers previously designed to amplify both the genomic DNA and the mRNA transcript from paraffin-embedded tissue. These products have predicted sizes of 250 BP and 154 BP, respectively, but our results showed that PCR amplification only (without reverse transcription) unexpectedly generated the 154-BP product. Further investigation of the nature of this product demonstrated that it originated from the amplification of DNA, not RNA. We conclude that the 154-BP product generated by these primers cannot be exclusively considered as beta-actin RNA product and should not be used to assess successful extraction of RNA, to ascertain its integrity, or to normalize for the total amount of RNA assayed by RT-PCR from paraffin-embedded tissue.

scholarly journals A Self-Contained Portable Polymerase-Chain-Reaction System Integrated with Electromagnetic Mini-Actuators for Bi-Directional Fluid Transport

2011 ◽  
Vol 27 (3) ◽  
pp. 357-364
Author(s):  
B. T. Chia ◽  
S.-A. Yang ◽  
M.-Y. Cheng ◽  
C.-W. Lin ◽  
Y.-J. Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Fluid Transport ◽  
Point Of Care ◽  
Reaction System ◽  
Thermal Characteristics ◽  
Pcr Amplification ◽  
Chain Reaction ◽  
Rapid Temperature ◽  
Small Footprint ◽  
Polymerase Chain

ABSTRACTIn this paper, the development of a portable polymerase chain reaction (PCR) device is presented. Integrating electromagnetic mini-actuators for bi-directional fluid transport, the proposed device, whose dimension is 67mm × 66mm × 25mm, can be fully operated with a 5V DC voltage. The device consists of four major parts: A disposable channel chip in which PCR mixture is manipulated and reacted, a heater chip which generates different temperature zones for PCR reaction, a linear actuator array for pumping PCR mixture, and a circuit module for controlling and driving the system. The advantages of the device include the rapid temperature responses associated with continuous-flow-type PCR devices, as well as the programmable thermal cycling associated with chamber-type PCR devices. The thermal characteristics are measured and discussed. PCR amplification is successfully performed for the 122 bp segment of MCF-7/adr cell line. Due to its small footprint, this self-contained system potentially can be employed for point-of-care (POC) applications.

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